Fluorescence offers an ideal solution for detection and sensing applications, as readout signals of probes or sensors containing fluorescent molecules are done by light.1-4 The probe or sensor may be read by using a microscopy, a spectrometer, the naked eye, CCD detectors, and cameras either directly or through fiber optics. Responsive sensor molecules are interesting in their own merit, and in particularly as they are the cornerstone in responsive imaging and sensing technology.
Two different types of responsive fluorescent molecules relevant for the present invention can be found in the literature: fluorophores where the binding/release of an analyte changes the intrinsic properties of the fluorophores e.g. fluorescein10, 11 and HPTS12, and fluorophores where binding of an analyte to an appended binding motive turns on/off the fluorescence of the fluorophores.13-15 The latter can be realized appending a photoinduced electron transfer (PET) quenching16 motif to a good fluorophore9, 17.
When a PET responsive fluorescent molecule absorbs a photon, an excited electronic state is created and an electron transfer prevents the excited molecule from emitting a photon. Thus, the fluorescence of the dye is quenched. Binding of an analyte to the acceptor/donor PET group changes its nature blocks the possibility of PET-quenching of the fluorophore. As a result, the fluorescent reporter moiety responds e.g. to a change in analyte concentration of the surrounding medium. Because binding of the analyte to the acceptor/donor group cancels the PET-quenching process, the fluorescence properties of PET-based sensors change as the concentration of the analyte changes.
pH is still the most crucial parameter in both biological and medicinal research and in the biotech industry. Cells are grown in media containing pH-indicators, and in every part of a biotechnological production line, pH is monitored directly. Fluoroscein is today, despite poor photostability, one of the most used pH-responsive fluorescence dyes, either as a cheap native, carboxy- or amino derivative, or as the expensive extended SNARF-type dyes.
Triangulenium compounds have for long been target molecules for research, as characterisation of some triangulenium compounds shows that these compounds may have some interesting optical properties. Triangulenium compounds may be described as comprising tris(2,6-dimethoxyphenyl)-methylium, DMP3C+, as backbone. Two methoxy groups may be fused together or replaced by e.g. a nitrogen atom to form a 6-membered ring structure comprising the two carbon atoms to which the methoxy groups are attached. The other remaining methoxy groups may optionally also be fused together or replaced by another atom, e.g. a carbon, oxygen, sulphur, or nitrogen atom. Thus, one, two or three 6-membered ring structures may be formed. Depending on the number of ring structures and the choice of the replacing atoms, different triangulenium compounds may be formed, amongst other TMA, ADOTA, DAOTA, and TATA
WO2005012436 discloses fluorescent dyes compounds belonging to the ADOTA subgroup. These compounds are suitable for use in visualisation of chemical compounds in e.g. chromatographic separation. The disclosed compounds contain at least two secondary or tertiary amine groups, which each are attached to a phenyl group present in the ADOTA subgroup. Further, the disclosed compounds also contain a functional group, which according to WO2005012436, allows the fluorescent dye to be attached.
Hamacek et at, Dalto Trans, 2012, 41, 6777-6782, discloses substituted compounds of the ADOTA, DAOTA, TATA subgroups. It is suggested to use these compounds for complexing with metallic cations. The luminescence and phosphorescence characteristics of the bonded metallic cation, Eu3+, are discussed.
Laursen, B. et at, Chem. Eur. J. 2001, No. 8, 1773-1783, discloses substituted compounds of the ADOTA, DAOTA, TATA subgroups. The absorption spectra of these compounds in their neutral and protonated form are discussed.
Hammerhoej et at, J. Org. Chem. 2012, 77, 5606-5612, discloses substituted aza-triangulenium fluorescent properties. The absorption and emission spectra of these compounds are discussed.
Research for developing new aza-triangulenium fluorescent dyes having improved photophysical properties is continuously ongoing. Accordingly, it would therefore be advantageously to find new fluorescent dyes having altered or improved photophysical properties, such as improved sensitivity and higher stability. Such compounds would be useful in sensors for monitoring or determining analytes. Therefore, the aim of the present invention is to provide improved fluorescent dyes having improved sensitivity, broad versatility as to range of analytes, large dynamic range, and/or photostability during use.